Studies of carbohydrate fermentation by Fusobacteria led to the discovery of a novel disaccharide phosphate hydrolase in F.mortiferum. This unique phospho-alpha-glucosidase (GlvA) hydrolyzed many phosphorylated compounds (including maltose-6- phosphate) and required divalent metal metal ion and nucleotide (NAD) for activity. In the past year, the same enzyme from Bacillus subtilis has been cloned and purified from a high expression system (Thompson et al. 1998 J. Biol. Chem.273:27347). The protein has now been crystallized. The thin rod-like crystals diffract beyond 2.2 Angstroms using synchrotron radiation, and a preliminary X-ray structure has been reported (Varrot et al. 1999 Acta Cryst. D55: 1212). Site-directed mutagenesis has allowed tentative identification of the beta-alpha-beta fold of the nucleotide-binding domain and the two catalytic glutamyl residues of the active site of the enzyme.A related, but stereochemically distinct, phospho- beta-glucosidase (CelF) has also been identified, cloned and purified from Escherichia coli.This enzyme hydrolyzes cellobiose-6-phosphate and, like the Bacillus enzyme, CelF also requires metal ion and NAD for activity. These novel bacterial enzymes are assigned to Family 4 of glycosylhydrolases. - PHOSPHO-ALPHA-GLUCOSIDASE, BACILLUS SUBTILIS, CELF, ESCHERICHIA COLI, FAMILY 4 OF GLYCOSYLHYDROLASES